Automated system for performing sample measurement, dilutions and photometric measurements

ABSTRACT

An automatic diluter and photometric reader adapted for use in the turbidimetric microbiological assays of antibiotics, vitamins, and the like, as well as in various other analytical assays.

United States Patent Kuzel et al. 1 Jan. 23, 1973 [S4] AUTOMATED SYSTEMFOR PERFORMING SAMPLE [56] References Cited MEASUREMENT, DILUTIONS ANDUNITED TATE P TENT PHOTOMETRIC MEASUREMENTS S S A S r a 3,567.390 3 1971R m 1 ..73/421 RX [75] Inventors: Norbert R. 'Kuzel, Ind1anapol1s,3,467,500 9/1969 f gz at a] Ind.; Frederick W. Kavanagh, 111- 3,567,3893/1971 Coulter et al. ..73/42l R X dianapo11s,Ind. 3,567,398 3/1971 Farr..23/259 [73] Assignee: Eli filly andCompanyJndianapolis, primaryExaminer Louis capozi .3 v. Assistant Examiner-.loseph W. Roskos [22] id; March 10 1 7 Attorney--Everet F. Smith and Houston L. Swenson [21]Appl.N0.: 122,843 57 ABSTRACT Related U.S. Application Data An automaticdiluter and photometric reader adapted [62] ofs N Sol 369 Feb 24 969 PmNo for use in the turbidimetric microbiological assays of 3 2 62 6antibiotics, vitamins, and the like, as well as in various otheranalytical assays.

l52| U.S. Cl. ..73/42l R, 23/259, 356/36 8 Chum", 5 nmwlng Flam-cs [51]Int. Cl. ..G0ln 1/10 Field of Search...73/42l R; 356/36; 23/253, 259

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mm kmzdo INVENTORS NORBERT R KUZEL FREDERICK W. KAVANAGH m mwH .wm Ci mmmv ATTORNEY PATENTEDJAN 23 I975 3.712.144

SHEET 2 or 2 INVENTORS N NORBERT R. KUZEL IO'OFREDERICK w. KAVANAGH H 4ICJKM m ATTORNEY AUTOMATED SYSTEM FOR PERFORMING SAMPLE MEASUREMENT,DILUTIONS AND PHOTOMETRIC MEASUREMENTS CROSS-REFERENCE TO RELATEDAPPLICATION This application is a division of our co-pending U. S.application, Ser. No. 801,369, filed on Feb. 24, 1969 and issued onSept. 28', 1971, as US. Pat. No. 3,609,040.

BACKGROUND OF THE INVENTION This invention relates to a two-componentsystem for performing turbidimetric microbiological assays on substancessuch as antibiotics, vitamins, and other bacterial growth promoting orinhibiting substances, as well as various other analytical assays, andthe like.

Turbidimetric microbiological assays of antibiotics, vitamins, and thelike have long been used as a means for identifying and assaying thepotency of such substances. The assay procedures are time consuming andusually involve many manual operations, each subject to some degree oferror.

In recent years, there have been attempts to provide automated devicesfor carrying out such assays. While there are currently several suchdevices, they are plagued with various problems such ascross-contamination between samples, slow rate of analysis, requirementsfor large volumes of sample, fixed time of incubation or reaction, lackof versatility, need for method changes to accommodate the equipment,etc. Furthermore, because of the cross-contamination problems of some ofthe currently existing devices, only one antibiotic, vitamin or the likecan be analyzed in a single operation. Before a second substance can beassayed, the equipment must be cleaned.

A further disadvantage of currently available equipment is their lack ofreproducibility in assays utilizing rod-shaped organisms such asKlebsiella, Lactobacillus, Pseudomonas, etc., because of the problem offlow birefringence (Kavanagh, F. W., Analytical Microbiology, Chapter 4,Academic Press, N.Y., N.Y. 1963).

In addition to the above disadvantages, the cost of many of thecurrently available devices is prohibitive.

Thus, while attempts have been made to provide automated devices forperforming turbidimetric microbiological assays, such attempts have beenfraught with problems.

SUMMARY OF THE INVENTION The two-component assay system of thisinvention comprises an automatic diluter module and an automaticphotometric reader-module. The modules perform the same basic operationsinvolved in traditional manual analytical or microbiologicalturbidimetric methods such as pipetting, diluting, and the like, withfar greater accuracy, precision, and efficiency. The modules, forexample, can utilize the same organisms, the same media, and can measurethe same bacteriological response employed in manual turbidimetricmicrobiological assays. The system is able to accommodate similar. assaydesigns and standardization procedures as have been used heretofore. Itprovides for unlimited incubation or reaction times.

Generally speaking, the assay system of this invention consists of twomodules, a diluter module, and a photometric reader module.

The diluter module includes a programmed control means, and a samplertray which is adapted to advance at a predetermined time, making eachsample tube available to a first sample probe means. A portion of thesample is withdrawn by a first sample probe means into a first meteringmeans having a zero dead-volume. The combined operations of the probeand metering means performs the pipetting operation. After the samplehas been measured, a predetermined amount of diluent is directed throughthe metering means by a first diluent delivery means, thereby washingout and diluting the sample into a diluted sample receiving means.

The operations are repeated until the desired number of samples havebeen measured and diluted. So long as the same diluent is used,different substances can be run by different methods without cleaningthe equipment between substances. The diluter module is self-cleaning.That is, by carefully choosing the materials of construction, and byproviding a sufficiently great dilution factor, the diluent serves tocompletely flush the sample from the metering means, thereby eliminatingcross-contamination without necessitating an intermediate cleaningoperation between samples.

A variety of dilutions can be obtained by either changing the volume ofthe diluent delivered or by altering the volume of sample measured bythe metering means.

The diluter module can additionally include an automatic means forconveying the diluted sample receiving means to a delivery tube which iscooperatively associated with the metering means.

The diluted samples can be transferred in suitable carriers, afterallowing for sufficient incubation or reaction time, to a photometricreader, preferably to the reader module of this invention.

The reader module of this invention includes a photometric reader suchas a photometer having a quartz flow cell as the cuvette. The readermodule has an automatic sample conveying means, such as a conventionallinear fraction collector or the like, cooperatively associatedtherewith for conveying each sample to be read to a means fortransferring the sample into the flow cell. The sample conveying meanscan advantageously be a probe means, or the like, which is adapted towithdraw a sample and convey it to the flow cell. Before each samplereaches the flow cell, the flow is momentarily interrupted to provide anair hammer" BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspectiveview of the diluter module.

FIG. 2 is a rear plan view of the probe assembly with the housingtherefor broken away.

FIG. 3 is a flow diagram of a metering cell in its first position.

FIG. 4 is a flow diagram of a metering cell in the second position.

FIG. 5 is a perspective view of the reader module with the position ofthe flow cell designated by dotted lines.

DESCRIPTION OF PREFERRED EMBODIMENTS Referring to the drawing, oneembodiment of the diluter module is shown generally at in FIG. 1. Thediluter module performs the functions of pipetting and delivering aprecise, predetermined amount of sample into a sample holder and ofadding a precisely measured amount of diluent or reagent thereto.

The diluter module includes a housing 11 for a programmed control unitand a drive mechanism therefor (not shown). The control unit and drivemechanism are actuated by control switches 12, and the main power switch13. The samples and standards are poured into sample tubes 14 which arethen placed into the sample holder 15. While any suitable sample holdercan be utilized, the preferred sample holder 15 includes three circular,spaced-apart plates. The top plate 16 has a centrally located apertureadapted to have a drive shaft 17 removably journalled therein, aplurality of spacedapart locating apertures 18 forming a concentriccircle about said centrally located aperture, said apertures beingadapted to receive a positioning pin 19 which is coupled to a mountingand supporting plate (not shown) and which limits the rotational motionof the sample holder so that each sample tube will be aligned with thesampler probe 32, and a plurality of spacedapart apertures 21 forreceiving sample tubes 14, the apertures being conveniently spaced apartadjacent the outer periphery of the plate. The center plate 22 has acentral aperture of sufficient size to fit over the housing 23 for thedriving motor which controls the indexing of the sample holder. Thedriving means in turn is controlled by the control unit. Apertures 24 inplate 22 are aligned with apertures 21 of the top plate 16. The bottomplate 25 similarly has a central aperture of sufficient size to fit overhousing 23. The bottom plate serves as a base for the bottom surface ofthe sample tubes. The three plates are maintained in their spacedapartpositions by spacing posts 26.

The samples are drawn into a first metering valve 30 via probe assemblyshown generally at 31. Probe assembly 31 includes a probe 32 which isremovably mounted at its upper end to a first mounting plate 33 viabracket means 34. A retaining means 35 can conveniently be fitted overthe top end of the probe to insure a secure fit into the bracket means.The probe is preferably of stainless steel or a like material. The probeis adapted to move vertically along channel 36 in probe assembly housing37. As can be seen in FIG. 2, a

second plate 38. is cooperatively associated with plate 33 so that plate38 is disposed adjacent the interior surface 39 of the front wall 40 ofthe probe assembly housing. Bearings 41 and 42 are rotatably retainedbetween plates 33 and 38. As the probe assembly moves along channel 32,bearings 41 and 42 ride along bearing runners 43. Drive shaft 44 iscoupled to and driven by, for example, a bell crank and motor (notshown).

When the probe assembly is actuated, the probe moves downwardly and intothe sample contained in the particular tube which is presently alignedwith the probe. A vacuum system (not shown) is then actuated and sampleis drawn through the probe and into a first loop 45 of a first meteringvalve means 30, via sample inlet tube 46. The vacuum system is then shutoff whereupon the loop remains full. The size of the sample which isultimately delivered into sample receiving means 47 via delivery tube 48is determined by the volume of the loop 45.

Once the sample is drawn into a first loop 45, and the vacuum is shutoff via vacuum solenoid (not shown), the sample is delivered viadelivery tube 48, into sample receiving means 47, which can be, forexample, a test tube, as a predetermined amount of diluent is passedinto the valve and through loop 45, through the valve, and into thesample retaining means via delivery tube 48. The presently preferredratio of diluent to sample is at least 10:1 to insure complete flushingof the valve, thereby eliminating cross-contamination. However, it isonly necessary that sufficient diluent be passed through the valve toflush out sample. Thus, for example, when large volumes of diluent arereceived, part of the diluent can be passed directly into the samplereceiving means. On the next cycle, the same sample is passed throughand measured by the second loop 49, which can differ in volume from thefirst loop 45 and is again washed out with diluent via delivery tube 48and into another sample receiving means.

Any unit capable of adding a specified amount of diluent can be utilizedin the practice of this invention, for example, the filling unitmanufactured by National Instruments Co. Diluent is pumped from thefilling unit 51 into the valve, via diluent inlet tube 52.

In the preferred embodiment of this invention, two metering valves 30and 30' are utilized in order to obtain, for example, two O.l5 ml.samples and two 0.1 ml. samples. The valves are conveniently mounted ona first surface of mounting board 53, which is, in turn, convenientlycoupled to housing 11 via support member 54 and mounting brackets 55.

The sample injection valve manufactured by Chromatonix Inc. isparticularly suitable for providing the sample metering means of thepresent invention when modified to operate in accord with the abovediscussion. However, any other suitable metering means can be utilized.

The operation of the valve 30 can be more fully understood by thediagram of FIG. 3. The sample is drawn into a first section 56 of valve30 via sample probe 32. The sample follows flow passage 57 to loop 45,and is drawn into a second section 56 of the valves, through passage 64,through vacuum valve 65 and to waste. When the vacuum valve is shut off,loop 45 remains filled until sections 56 and 56' are switched to theiralternate positions as shown in FIG. 4. The diluent then passes throughpassage 58, into loop 45, thereby washing out the sample via passage 60and through the delivery tube to the awaiting sample receiving means.

Concurrently with the diluent delivery through loop 45, loop 49 isconnected to the sample probe via passage 61 and 62, thereby fillingloop 49 with the same sample. Return of 56 and 56' to the first positionnow connects loop 49 with the diluent via passage 63 and 66.

The valves are preferably of Teflon and Kel-F, although other suitablematerials can be employed. Similarily, the loops are preferably Teflon.

The sample receiving means 47 are conveniently held in racks 82. Theracks are then conveniently advanced automatically into the properposition by an automatic conveyor means 83, cooperatively associatedwith housing 11 and mounting board 53 so that for each delivery cycle, asample receiving means is aligned beneath a delivery tube.

It can be seen that the diluter module provides a convenient, accuratemeans for automatically pipetting a given amount of sample, deliveringthe sample to a sample holder such as a test tube or the like, andadding.

thereto, a predetermined amount of medium, diluent, reagent, or thelike.

Referring now to FIG. 5, the reader module of this invention is showngenerally at 70. The diluted samples obtained from the diluter module,or any other suitable means, are, after proper incubation or reactiontime, transferred in the racks 82 to an automatic conveyor means 83, orother suitable advancing means. It can be seen that the racks are guidedby guide means 71, and that a retaining plate 72 is positioned behindthe last rack. The samples are drawn into a suitable photometer 73 byprobe assembly 31. The samples are passed through flow cell 74 via inlettube 75 and through outlet tube 76 to a waste receiving means (notshown). The flow cell is preferably quartz or a like material, andreplaces the cuvette of conventional readers. A solenoid valve (notshown) in the control unit 79 controls the sample flow. The controlcircuit is adapted to momentarily drop out the solenoid and interruptthe sample flow before it reaches the flow cell. This produces an airhammer effect which takes care of any air bubbles present in the flowstream. It is presently preferred, when determining the turbidity ofrodshaped organisms, that the flow cell permit a rate of flow of notless than 0.8 ml./sec., and preferably a rate of l ml./sec. or higher inorder to obtain highly accurate, reproducible results. For other assays,the flow rate is not critical. The readings can be taken from the scale77 of the photometer 73, or, as in the preferred embodiment, a suitablereadout device 78 can be cooperatively associated with the photometer.

The readout is compatible with a variety of data acquisition devicessuch as printed tape, punched paper tape, punched cards, or can beinterfaced directly to a computer.

The reader module of this invention provides a great advance in the artby providing a means for overcoming the problems of measuring theturbidity of rod-shaped organisms by avoiding the flow birefringenceproblem normally encountered with rod-shaped particles. By producing asufficiently rapid flow through the flow cell, rod-shaped particles,which would otherwise orient themselves with the slow currents in astatic cell, leading to inconsistent readings, are read with the sameprecision as are spherical particles. Thus the present reader module nowmakes it possible to assay for substances which affect variousrod-shaped organisms such as Klebsiella, Lactobacillus, Pseudomonas, andthe like, and to determine them with a precision equivalent to sphericalorganisms, all other factors being equal.

While the present invention is particularly suited for turbidimetricmicrobiological assays, it will be apparent to those skilled in the artthat the diluter module and the reader module can be used ointly orseparately in any number of analytical procedures, medical laboratorytests, and the like. Therefore, while the following discussion isdirected toward turbidimetric microbiological assays, the modules ofthis invention are applicable to any suitable analytical procedures.

We claim:

1. In an automatic dilutor module the improvement comprising: a valvemeans having first and second sections, said first section having adiluent supply inlet connected to a metered diluent supply and a samplesupply inlet connected to a sample supply, a pair of loops ofpredetermined volume having their first ends connected to a pair ofrespective outlets in said valve means first section in alternatelyselective communication with said diluent supply and said sample supply,said loops having their second ends in alternately selectivecommunication with a pair of respective discharge outlets in said secondsection of said valve means, said pair of discharge outlets in saidsecond section of said valve means being in communication with a wastereceptacle and a delivery receptacle, and control means associated withsaid first and second sections alternately effecting coupling of each ofsaid loops between (a) said diluent supply inlet and said deliveryreceptacle; and (b) said sample supply inlet and said waste receptacle.

2. In an automatic dilutor module the improvement in accordance withclaim 1 in which said loops are interchangeably mounted.

3. In an automatic dilutor module the improvement in accordance withclaim 2 in which the coupling of one loop between said diluent supplyinlet and said delivery receptacle is concurrent with the coupling ofthe other loop between said sample supply inlet and said wastereceptacle.

4. In an automatic dilutor module the improvement in accordance withclaim 3 in which the communication betweensaid sample supply inlet andsaid sample supply includes a probe extending into said sample supply.

5. In an automatic dilutor module the improvement in accordance withclaim 4 in which said sample supply is contained in a plurality ofopen-mouth receptacles.

6. In an automatic dilutor module the improvement in accordance withclaim 5 in which said probe is reciprocably mounted and associated witha drive means for imparting vertical movement thereto.

7. ln an automatic dilutor module the improvement in accordance withclaim 6 in which said open-mouth receptacles containing said samplesupply are automatically driven in succession under said probe and insynchronization with 'the vertical movement of said probe.

8. In an automatic dilutor module the improvement in accordance withclaim 7 in which a plurality of delivery receptacles are automaticallydriven in successive synchronized communication with the discharge ofdiluted samples from the one of said discharge outlets.

1. In an automatic dilutor module the improvement comprising: a valvemeans having first and second sections, said first section having adiluent supply inlet connected to a metered diluent supply and a samplesupply inlet connected to a sample supply, a pair of loops ofpredetermined volume having their first ends connected to a pair ofrespective outlets in said valve means first section in alternatelyselective communication with said diluent supply and said sample supply,said loops having their second ends in alternately selectivecommunication with a pair of respective discharge outlets in said secondsection of said valve means, said pair of discharge outlets in saidsecond section of said valve means being in communication with a wastereceptacle and a delivery receptacle, and control means associated withsaid first and second sections alternately effecting coupling of each ofsaid loops between (a) said diluent supply inlet and said deliveryreceptacle; and (b) said sample supply inlet and said waste receptacle.2. In an automatic dilutor module the improvement in accordance withclaim 1 in which said loops are interchangeably mounted.
 3. In anautomatic dilutor module the improvement in accordance with claim 2 inwhich the coupling of one loop between said diluent supply inlet andsaid delivery receptacle is concurrent with the coupling of the otherloop between said sample supply inlet and said waste receptacle.
 4. Inan automatic dilutor module the improvement in accordance with claim 3in which the communication between said sample supply inlet and saidsample supply includes a probe extending into said sample supply.
 5. Inan automatic dilutor module the improvement in accordance with claim 4in which said sample supply is contained in a plurality of open-mouthreceptacles.
 6. In an automatic dilutor module the improvement inaccordance with claim 5 in which said probe is reciprocably mounted andassociated with a drive means for imparting vertical movement thereto.7. In an automatic dilutor module the improvement in accordance withclaim 6 in which said open-mouth receptacles containing said samplesupply are automatically driven in succession under said probe and insynchronization with the vertical movement of said probe.
 8. In anautomatic dilutor module the improvement in accordance with claim 7 inwhich a plurality of delivery receptacles are automaticalLy driven insuccessive synchronized communication with the discharge of dilutedsamples from the one of said discharge outlets.